Objective biochemical markers of sperm maturity and function

(Relationship between sperm maturity, fertility and membrane properties)

Sperm-hemizona

complex

Only the clear headed sperm, which have completed cytoplasmic extrusion and

plasma membrane remodeling (proven), were able to bind to the zona pellucida.

Huszar et al., Mol Reprod Dev 1993; Fertil Steril,1994; BOR, 1997

CK immunostaining

Huszar et al. BOR, 2000

Spermiogenesis: Sperm with Normal

and Diminished Development Mature

Development Development

DNA Nick Translation Aniline blue CK Sperm field pairs treated by various biomarkers (semen and HA-selected sperm)

semen

HA selected

CK-immuno Persistent histones DNA chain fragmentation

Huszar et al., 2007

Acridin orange treated spermatozoa in semen and in HA-selected sperm

fraction (red sperm: DNA fragmentation, green sperm: high DNA integrity)

Yagci and

HUSZAR, 2010

PICSI SPERM SELECTION

(Jakab et al., F+S, 2005)

Clinical Loss Rate

0.067 0.011

< 0.001

KCWorrilow et al, 2013

Sperm Selection Methods

Except HA-selected sperm, little research on sperm attributes, such

as chromatin, DNA integrity, and chromosomal aneuploidies.

Hyaluronic acid-mediated sperm selection

Density gradient

IMSI

Microfluidics

Electrophoretic separation

Birefrigence (polarized light)

Raman spectroscopy (single sperm too, recent

paper-ZP-bound sperm)

PROPERTIES OF HYALURONIC ACID-BOUND SPERM

EXCELLENT SPERM VIABILITY

NO SURPLUS CYTOPLASMIC CONTENT

KRUGER NORMAL MORPHOLOGY

NO PERSISTENT HISTONES, thus normal histone-protamin progression (DNA PACKING)

HIGH DNA CHAIN INTEGRITY, NO FRAGMENTATION (THREE STUDIES shown)

NO APOPTOTIC PROCESSES

NORMAL FRQUENCY OF CHROMOSOMAL ANEUPLOIDIES

CURRENT ONGOING: RNA STUDIES, EPIGENETIC STUDIES

(Wish list of the IVF-ICSI Profession)

In all aspects studied zona pellucida and HA-bound sperm are identical

Destaining

FISH

Nick Translation

Caspase-3 Immunostaining

Localization

+ Imaging

Re-localization

+ Imaging

Preparation, Fixation

Aniline Blue staining

of persistent histones

SATI, L., et al., RBMonline, 2008

Aniline blue staining pattern and DNA fragmentation

(in situ nick translation)